We will characterize two signaling systems that regulate the permeability and integrity of blood vessels: coagulation proteases and protease-activated receptors, and sphingosine-1-phosphate and S1P receptors. We will test the hypothesis that both systems sense extravasation of plasma and trigger appropriate endothelial cell responses, and we will explore parallels and possible connections between these systems. We shall ask: 1) How is S1P important for regulation of vascular permeability and integrity? We generated adult mice that fail to supply S1P to plasma and found striking abnormalities in vascular permeability and integrity. We shall determine a) whether altered barrier function in these "pS1Pless" mice is due to failure to metabolize sphingosine with consequent endothelial cell-autonomous metabolic/toxic effects or to failure to supply S1P to plasma and S1P receptor activation on endothelial and other cells, b) the anatomic basis for their increased vascular leak and whether it shows tissue or vessel-type specificity, c) whether endothelial cells are the main target of plasma S1P signaling in this context, and if so, whether such signaling is continuous or whether plasma S1P provides a dynamic signal that enables endothelial cells to sense and help terminate leaks, and d) the long-term effects of lack of plasma S1P and whether they are due to dysregulated barrier function. 2) How are PARs important for regulation of vascular permeability and integrity? We generated knockout mice for all the PARs and relevant transgenics and will use these to determine a) the effects of activation of different endothelial PARs on vascular permeability and integrity in vivo and whether pS1Pless mice provide a sensitized system for uncovering such roles for PARs, b) whether PAR signaling is parallel to, partially redundant with, or dependent upon S1P signaling. 3) Do differences in apical and basal S1P and PAR function contribute to their roles in barrier regulation? Our preliminary studies suggest a model that would permit S1P signaling to serve a dynamic leak detector function and raise new questions regarding PARs by analogy. We will determine a) whether endothelial cell S1P1 receptors display apical-basal polarity in vitro and in vivo to enable the dynamic leak detecting function we posit, and b) whether apical and basal differences, such as EPCR ligation, modulate the effects of PAR activation on either surface. Complementary genetic and pharmacological approaches will be used in mouse models and in cell culture. Preliminary studies reveal an important role for plasma S1P in regulating endothelial barrier function in vivo, distinct barrier responses to activation of different PARs, dramatic effects of manipulation of S1P and PAR signaling on survival in models of anaphylaxis, and long-term effects of altered barrier function in vivo. The proposed studies will provide new information regarding vascular physiology and pathophysiology. PUBLIC HEALTH RELEVANCE: Our studies will reveal new molecular and cellular mechanisms by which the endothelial cells that line blood vessels sense and regulate leakiness. Alterations in endothelial permeability play an important role in allergic reactions, new blood vessel growth in cancer, heart attacks and strokes, atherosclerosis, and inflammatory states including sepsis. Thus, understanding of how to manipulate the endothelial leakiness might benefit patients with a range of disorders.